Noninvasive detection of colonic biomarkers using fecal messenger RNA

ABSTRACT

A noninvasive method utilizing feces, containing sloughed colonocytes, as a sensitive technique for detecting diagnostic colonic biomarkers as well as a method for isolating poly A +RNA from feces. The method allows the isolation and quantitation of specific eukarotic mRNAs as candidate biomarkers for colon cancer isolated from feces.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application was filed under 37 CFR 371 from PCT Application No.PCT/US98/06698 filed Apr. 3, 1998, which claims priority of U.S.Provisional Patent Application Ser. No. 60/043,048, filed Apr. 4, 1997,both being incorporated herein by reference, is hereby claimed.

S

TATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT Notapplicable

REFERENCE TO A “MICROFICHE APPENDIX”

Not applicable

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to methods for the noninvasive detectionof colonic biomarkers using fecal messenger RNA (mRNA). Moreparticularly, the present invention relates to methods for the isolationof poly A+ RNA from feces, and includes the subsequent detection of, andquantitation of, particular mRNAs that correlate with a patient'sdiagnosis and/or prognosis of colon cancer thereby providing methods fornoninvasively diagnosing and/or prognosticating colon cancer in apatient. One embodiment of the present invention relates to thedetection of, and quantitation of, mRNA from sloughed colon cells infeces encoding particular isozymes of protein kinase C (PKC) whoselevels are correlative with and predictive of colon cancer in a patient.Methods including semi-quantitative RT-PCR and biochip microarraytechnology may be made to assay and evaluate the fecal poly A+RNA.

2. General Background

Since colon cancer is the second most common cause of U.S. cancer deathsand since early detection can result in a high cure rate, an accuratescreening method for colon cancer is imperative. Current detectionmethods have many drawbacks. For example, fecal occult blood screeningcan produce false positive results due to meat consumption, ironsupplement intake and other common behaviors. The other routinescreening technique, sigmoidoscopy, is an invasive expensive procedurewhich has inherent risks of perforation, reaction to sedative, orbleeding. In addition, the efficacy of sigmoidoscopy screening remainsunproven (Levin, 1996). Because of these limitations, colon cancer curerates have not improved in the past 30 year (Silverberg, 1988, WFR/AICR,1997). Therefore, an accurate technique to detect early changesassociated with the tumorigenic process is imperative in order todecrease the mortality from colon cancer.

Screening of colorectal cancer is recommended for all persons aged 50and older with annual fecal occult blood testing or sigmoidoscopy, orboth (Levin, 1996). However, each of these tests has limitations relatedto sensitivity and specificity (Levin, 1996). The presence of colorectaland pancreatic tumors has been detected in the stool and coloniceffluent of patients by noninvasive methods based on the molecularpathogenesis of the disease (Sidransky, 1992: Tobi, 1994; Caldas, 1994).These protocols utilize DNA extraction procedures and the detection ofoncogene mutations using PCR. The major disadvantage of this methodologyis that it will not detect alterations in gene expression. Ourmethodology can quantitate the expression of any relevant gene byisolating and amplifying mRNA derived from fecal material containingsloughed colonocytes.

A sensitive molecular technique for the detection of colon cancer isimportant since early diagnosis can substantially reduce mortality(Levin, 1996). Our method is noninvasive, highly sensitive and specific.Our protocol is unique because it will determine colonic expression ofany gene (e.g., tumor suppressor gene, oncogene), and provides earlysensitive prognostic information and greatly enhances current methods ofdietary and pharmacologic risk assessment.

SUMMARY OF THE PRESENT INVENTION

The present invention relates to a novel non-invasive technology todetect changes in colonocyte gene expression associated with earlystages of colon tumorigenesis. This invention also covers the firstknown methods to isolate poly A+ RNA from feces. This methodology hasthe advantage of utilizing a fecal sample, which contains sloughed coloncells. Therefore, it does not require anesthesia or cause any discomfortto the patient. In addition, the invention utilizes a novel mRNAisolation process that results in an unexpectedly high yield andstability of isolated fecal mRNA, and utilizes an exquisitely sensitivetechnique, rapid competitive polymerase chain reaction (Jiang, 1996),developed by the inventors, to detect and quantify mRNA markers of thetumorigenic process. Thousands of gene markers for the tumorigenicprocess are assayable in the practice of the present invention. Thesemarkers include, but are not limited to, PKC isozymes such as, forexample, PKC βII (PKC beta II) and PKC ζ (PKC zeta), where, for example,levels of these particular isozymes in feces are correlative of andpredictive of the presence of, and development of colon cancer in a ratcolon cancer model (Davidson, 1998). We have also successfully isolatedpoly A+ RNA from rectal vault eluate isolated at the initiation ofcolonoscopy. Yields from fecal eluate are generally in the range of0.3-1.5 μg poly A+ RNA isolated per subject.

The pathogenesis of colon cancer is a multi-step process, in which tumorsuppressor genes, oncogenes and other molecules involved in signaltransduction are affected (Fearon, 1997). It is now clear the signalsmediated via select isozymes of protein kinase C (PKC) are involved incolonic tumor development (Sakanoue et al., 1991; Kopp et al. 1991; Baumet al., 1990). PKCs are a family of serine-threonine kinases thought toregulate colonic cell proliferation, differentiation and programmed celldeath. PKCs can be divided into three different sub-categories based onthe cofactors needed for activation: classical PKCs (α, βI, βII and γ)require diacylglycerol (DAG) and Ca²⁺for activation; novel PKCs (δ, θ, ηand ε) are Ca²⁺independent, but activated by DAG; and atypical PKCs (λ,ι and ζ) are Ca²⁺and DAG independent. Although these isozymes areenzymatically similar, in vivo, they have different expression patternsdepending on tissue and cell type (Blobe et al., 1996).

PKC βII protein is generally found in very low levels in normal ratcolonic mucosa (Davidson et al., 1994). However, βII protein levelsincrease in colonic tumors as compared with normal colonic mucosa(Craven et al., 1992; Wali et al., 1995). In contrast, PKC ζ mRNA levelsare significantly lower in human colorectal tumors than in normalcolonic mucosa (Kuranami et al., 1995). PKC ζ protein levels also aresignificantly lower in preneoplastic colonic epithelium from ratsinjected with azoxymethane (AOM) as compared with saline-injectedcontrol rats (Wali et al., 1995; Roy et al., 1995; Jiang et al., 1997).Therefore PKC βII and ζ may serve as biomarkers to monitor thedevelopment of colon cancer.

In summary, no one has reported the isolation of intact poly A+ RNA fromfecal material or rectal eluates obtained at colonoscopy. Utilization ofthis noninvasive procedure combined with either RT-PCR analyses orgenechip microarrays is novel.

BRIEF DESCRIPTION OF THE DRAWINGS

For a further understanding of the nature and objects of the presentinvention, reference should be had to the following detailed descriptiontaken in conjunction with the accompanying drawings, in which like partsare given like reference numerals and wherein:

FIG. 1 shows representative competitive PCR products for determinationof Liver-Fatty Acid Binding Protein (L-FABP) expression in fecal poly A+RNA. Lane 1, marker; lane 2, rat colonic mucosa (standard); lanes 3-6,rat poly A+ RNA. Upper band is amplified sample band (390 bp); lowerband is amplified internal standard (336 bp).

FIG. 2 shows a representative gel of RC-PCR products of PKC βII. Lane 1,marker; lanes 2-5 fecal poly A+ samples. Upper band is the amplifiedsample band (419 bp); the lower band is the amplified internal standard(361 bp).

FIG. 3 shows a representative gel of RT-PCR products of PKC βI and PKCγin brain but not in fecal poly A+ RNA. Lane 1, marker; lane 2, PKC βI inbrain (639 bp); lanes 3 and 4, PKC βI in fecal poly A+ RNA; lane 5, PKCγ in brain (347 bp); lanes 6 and 7, PKC γ in fecal poly A+ RNA.

FIG. 4 shows expression of PKC βII in fecal poly A+ RNA or colonicmucosal RNA. Rats were injected with azoxymethane (AOM) or saline twice.Feces were collected 36 weeks after the second injection and poly A+ RNAwas isolated. Colonic mucosa was scraped and total RNA was isolated.Quantitative RC-PCR was performed using primers specific for PKC βII.PCR products were separated on 4% agarose gels, stained with ethidiumbromide, photographed and scanned on a densitometer to quantitate.Y-axis represents band intensities (OD×mm²). (A) Expression of PKC βIIin fecal poly A+ RNA from rats with or without tumors (mean±SEM;P=0.026; n=12-29). (B) Expression of PKC βII in colonic mucosal RNA fromrats injected with AOM or saline (mean±SEM; P=0.036; n=16-20). “BI” is“band intensity”, “T” is “tumor”, “NT” is “no tumor”, “I” is“injection”, and “S” is “saline”.

FIG. 5 shows expression of PKC ζ in fecal poly A+ RNA from rats injectedwith AOM or saline. See FIG. 4 legend for further details (mean±SEM:P=0.017; n=21-22).

FIG. 6 shows expression of PKC βII/PKC ζ ratio in fecal poly A+ RNA fromrats with or without tumors. See FIG. 4 legend for further details(mean±SEM; P=0.025; n=9-26).

FIG. 7 shows a representative agarose gel of PKC βII (A) and PKC ζ (B)RT-PCR products from human rectal vault eluate obtained at theinitiation of colonoscopy and from freshly isolated human fecal poly A+RNA. Lanes 1 and 2, amplification from rectal vault eluate poly A+ RNA;lane 3, amplification from fecal poly A+ RNA; lane 4, minus RT negativecontrol; lane 5, amplification from human brain poly A+ RNA (positivecontrol); lane 6, base pair marker. PKC βII product is 280 bp, PKC ζproduct is 216 bp.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The development of noninvasive techniques, as shown in this invention,provides early sensitive prognostic information and will greatly enhancethe current methods of dietary, pharmacologic, and cancer riskassessment. The present invention describes a noninvasive methodutilizing feces containing sloughed colonocytes as a sensitive techniquefor detecting diagnostic biomarkers in the colon. By incorporating anovel method of isolating fecal mRNA and by utilizing the exquisitesensitivity of quantitative rapid competitive reverse transcriptasepolymerase chain reaction (RC-PCR), the method is capable of isolatingand quantitating specific messenger RNAs (mRNAs) as candidate biomarkersin feces. The RNA can also be assayed and evaluated using nucleic acid“biochip”/“microarray” technology as described below and as understoodin the art. This technology allows for large-scale high-throughputmonitoring of gene expression patterns of up to 40,000 genes (Lipshutz,1999). Further, the present invention has recognized a correlationbetween levels of particular biomarkers and the presence of anddevelopment of colon cancer.

For example, but not in a limiting sense, the present inventionrecognizes that PKC βII expression in fecal poly A+ RNA is positivelycorrelated with tumor incidence and the expression of PKC ζ isnegatively correlated with tumor incidence (Davidson, 1998).

The method of the present invention involves a novel technique ofisolating mRNA from feces that results in, inter alia, substantialimprovement in yield, and stability of isolated poly A+ RNA fromexfoliated colonocytes in feces, in a substantially reduced amount oftime compared with the only other known techniques in the art (thetechnique of Davidson, 1995).

Approximately one-sixth to one-third of normal adult colonic epithelialcells are shed daily (Potten, 1979). Isolation of colonocytes from feceshas been reported by another group (Albaugh, 1992). This method is verytime consuming and results in an extremely low yield such that usefuldiagnostic tests on the isolated cells are limited and very laborintensive. We therefore designed a technique to directly isolate poly A+RNA from feces containing exfoliated colonocytes. The poly A+ RNAisolated can be used to probe for early markers for colon cancer orother colorectal diseases.

Specifically, we have redesigned the protocol of the prior art (forexample, Davidson, 1995) to significantly simplify and enhance theprocess, resulting in a greatly enhanced yield. In addition, we havecombined the improved isolation protocol with an extremely sensitivedetection technique, called rapid competitive polymerase chain reaction(RC-PCR), a technology developed in our laboratory.

The original method (Davidson, 1995) involved the isolation of total RNAfrom feces followed by poly A+ RNA isolation, which could subsequentlybe utilized for assessment of colon cancer biomarkers. This oldermethodology resulted in a relatively low yield of poly A+RNA, therebylimiting the diagnostic tests which could be performed. Themodifications, detailed below, result in approximately 10-fold increasein poly A+ RNA yield, allowing for extensive screening of various coloncancer biomarkers. In addition, the method is straight-forward and couldbe performed by a trained technician. Several samples (up to 12 or more)can be processed at once.

The refined RNA isolation technology of the present invention has beenvalidated using the rat chemical carcinogen model. Specifically, we havedemonstrated that protein kinase C (PKC) βII and PKC ζ G in exfoliatedcolonocytes may serve as noninvasive markers for development of colontumors (Davidson, 1998).

The improved method is an improvement on the basic method set forth byLaurie A. Davidson, Yi-Hai Jiang, Joanne R. Lupton, and Robert S.Chapkin in Noninvasive Detection of Putative Biomarkers for Colon CancerUsing Fecal Messenger RNA, published in Cancer Epidemiology, Biomarkers& Prevention, Vol. 4, 643-647, Sept., 1995—this paper is herebyincorporated by reference in its entirety. Instant improvements include,for example, poly A+ RNA is directly isolated from feces using oligo dTcellulose based methodology. The previous published report (Davidson,1995) involved total RNA isolation from feces followed by poly A+isolation from the total RNA preparation. The improved method shortensthe poly A+ RNA isolation to 5 h (from 2 d with the previousmethodology) and significantly increases yield by 5-10 fold.

In still another feature of the present invention, and an improvementover the prior art, the present invention is suitable for the detection,and quantitation of specific biomarkers whose expression in colon cellsand thus, in poly A+ RNA isolated from feces, correlates with and ispredictive of states of colon cancer in a patient.

For example, the present invention shows that PKC βII and PKC ζ aresuitable as biomarkers for monitoring the development of colon cancer.The modulation of these putative biomarkers—affected by the presence orabsence of colon tumors is shown herein. Weanling rats were injectedwith saline (control) or carcinogen (azoxymethane). Fresh fecal samples(n=6 per diet) were collected 36 weeks post injection, poly A+ RNA wasisolated and quantitative RC-PCR performed using primers to PKC βII andζ. PKC isozyme expression was altered by the presence of tumors(P<0.05), with tumor bearing animals having a 3-fold higher βIIexpression and 6-fold lower ζ expression in exfoliated colonocytes thannon-tumor bearing animals. We propose that expression of PKC βII and ζin exfoliated colonocytes may serve as a noninvasive marker fordevelopment of colon tumors.

Also novel is the use of the rapid competitive PCR method (as firstdisclosed in Jiang, 1996) to sensitively quantify biomarker expressionin fecal poly A+ RNA. This method is described in detail in Rapidcompetitive PCR determination of relative gene expression in limitingtissue samples, Yi-Hai Jiang, Laurie A. Davidson, Joanne R. Lupton, andRobert S. Chapkin, Clinical Chemistry, 42:2, 227-231 (1996), which ishereby incorporated by reference in its entirety. This method is idealfor limited RNA samples, since it requires only a single PCR reaction inorder to determine relative gene expression. In contrast, the moretraditional mimic reverse transcriptase (RT)-PCR technique requires aseries of 5 to 7 PCR reactions in order to quantitate gene expression.

For example and for illustrative purposes only, at least the followingfeatures of the present invention are novel over the prior art: (1)Direct isolation of poly A+ RNA from feces or rectal eluates; (2)Ten-fold increase in poly A+ yield with decrease in processing time bymore than 50%; (3) Identification of protein kinase C (PKC) βII as amarker for colon cancer; (5) Use of the novel relative competitiveRC-PCR method to detect and quantify markers of colon cancer in fecescontaining exfoliated colon cells; and (6) Validation of fecalhomogenate stability after processing and storage prior to poly A+isolation.

The methods of the present invention can be utilized to detectpredictive risk markers for colon cancer including, but not limited to,biomarkers such as:

Acyl CoA Binding Protein (ACBP) expression

Arginase expression

bax expression

bcl-2 expression

Bcl-XL expression

Bcl-Xs expression

c-myc expression

Carcinoembryonic Antigen (CEA) and Nonspecific Crossreacting Antigen(NCA) expression

CD44 Glycoprotein expression

Cyclin-dependent kinase inhibitors (p27, p16ink4) expression

Cyclin-dependent kinase cdk2/cdc2, cyclin D1, and cdk4 expression

Cyclooxygenase I and II

Decay Activating Factor expression

E-Cadherin cell adhesion molecule expression

Epidermal Growth Factor Receptor (EGFR) expression

Fatty Acid Synthase expression

Fecal alpha-1 Antitrypsin expression

GDP-L-fucose:beta-D-galactoside-alpha-2-L-fucosyltranferase expression

Gluthathione S-Transferase expression

Histone H3 expression

Interleukin 1 and 2 expression

Liver and Intestinal Fatty Acid Binding Protein expression

hTERT expression

Mitogen-activated protein kinase (MAP kinase) expression

MAP kinase phosphatase-1 expression

NO synthase, inducible expression

Omithine Decarboxylase expression

p21 waf 1/cip 1 expression

P-glycoprotein, the mdr gene product expression

Plasminogen Activator expression

Proliferating cell nuclear antigen (PCNA) expression

Prostaglandin Synthase Type II (COX II) expression

Protein Kinase A, Type I and II Isozyme expression

Protein Kinase C α, βII , δ, ε, λ, ι, μ, ζ expression

Ras oncogene expression

Ras oncogene mutations

Stearoyl-CoA desaturase expression

Sterol Carrier Protein-2 (SCP-2) expression

Telomerase expression

Transforming Growth Factor-beta I and II expression

Transforming Growth Factor-beta type II Receptor expression andmutations

Tumor Necrosis Factor Alpha expression

Tumor suppressor gene APC mutations

Tumor suppressor gene p53 mutations and expression

Tumor suppressor gene retinoblastoma (Rb) protein expression

Villin expression 1,25-dihydroxyvitamin D3 Receptor expression, and13-hydroxyoctadecadienoic acid (13-HODE) dehydrogenase expression.

The present invention is suitable for noninvasive detection of anydiagnostic gene or panel of genes including PKC isozymes as predictiverisk markers for human colon cancer. We have already validated the useof select PKC isozymes as predictive risk markers using the ratexperimental colon cancer model (Davidson, 1998). In addition, we haveisolated human poly A+ RNA from feces and rectal eluates and detectedthe presence of PKC isozymes.

Additionally, the present invention, using, for example the rat coloncancer model, relates to the determination of the temporal effects ofcarcinogen on select PKC isozyme fecal mRNAs.

The development of noninvasive techniques, as shown in this invention,provide early sensitive prognostic information and greatly enhancecurrent methods of dietary and pharmacologic risk assessment. The methodreported herein is novel since it is the first to report that poly A+RNA from exfoliated colonocytes can be isolated directly from feces orrectal eluates and can be used to probe for markers of colon cancer.Several markers have also been identified that are present in fecal polyA+ RNA that predict for colon cancer.

EXPERIMENT 1

Utilization of Isolated Fecal Poly A+ RNA to Detect Colon Cancer Markers

Further details related to this method may be found in the article byLaurie A. Davidson, Christin M. Aymond, Yi-Hai Jiang, Nancy D. Turner,Joanne R. Lupton and Robert S. Chapkin, entitled “Non-invasive detectionof fecal protein kinase C βII and ζ messenger RNA: putative biomarkersfor colon cancer”, published in Carcinogenesis, vol. 19, no. 2, pp.253-257, 1998, which is hereby incorporated by reference in itsentirety.

Experimental Methods

Isolation of poly A+ RNA from feces:

1. Collect 0.3-2.0 g of rat or human feces. Within 30 min of defecation,add 10 volumes of Lysis Solution (available from Poly A+ Pure Kit,Ambion, Austin, Tex.) (“Ambion Kit”). Homogenize feces with a pestle.This homogenate can be stored at −80° C. for several months beforefurther processing.

2. Transfer homogenate to sterile 50 ml conical Falcon tube and measurethe volume. Add 2 vol Dilution Buffer (Ambion Kit). Mix by inversion for10 sec. Centrifuge at 4,000×g, 15 min, 4° C. Transfer supernatant to anew sterile 50 ml Falcon tube.

3. Add oligo dT cellulose (Ambion Kit), an amount equal to 10% of thestarting fecal weight. Mix by inversion to resuspend the oligo dT resin.

4. Rock the tube on a horizontal shaker at 100-150 rpm at roomtemperature for 1 hr.

5. Pellet the oligo dT resin by centrifuging at 4,000×g, 3 min, 4° C.Remove and discard the supernatant.

6. Resuspend the resin with 6-10 ml Binding Buffer (Ambion Kit) and mixwell. Pellet resin as described in step 5 and discard. Repeat this twomore times.

7. Resuspend resin with 6-10 ml Wash Buffer (Ambion Kit) and mix well.Centrifuge as described in step 5 and discard supernatant. Repeat thiswash two more times.

8. Resuspend the resin in 1-2 ml wash buffer and transfer to a spincolumn in a 1.5 ml microfuge tube (Ambion Kit). Centrifuge at 5,000×g,10 sec, room temperature to remove the supernatant. Place spin columninto a new microfuge tube.

9. Add 300 μl Elution Buffer (Ambion Kit) which has been pre-warmed to65° C. Immediately centrifuge at 5,000×g, room temperature, 30 sec andsave the eluate. Add another 300 μl pre-warmed Elution Buffer andcentrifuge at 5,000×g, room temperature, 30 sec. Combine eluate withprevious eluate. Discard the spin column.

10. Precipitate the poly A+ RNA by adding 60 μl 5M ammonium acetate, 10μg glycogen and 2.5 vol 100% ethanol. Place at −80° C. for 1 h. Recoverpoly A+ RNA by centrifugation at 12,000×g, 20 min, 4° C. Remove anddiscard supernatant, add 0.5 ml chilled 80% ethanol to the tube, inverttube gently. Resuspend the poly A+ pellet in 60-200 μl water/0.1 mMEDTA. Vortex gently to resuspend.

This purified A+ RNA is used for colon cancer biomarker studies such asthose detailed below:

Results

Using the method described above, fecal poly A+ RNA from rats injectedwith carcinogen or saline (control) was examined for colon cancerbiomarkers. We determined that protein kinase C βII expression in fecalpoly A+ RNA is positively correlated with colon tumor incidence (FIG.4A), while protein kinase C ζ is negatively correlated with tumorincidence (FIG. 5).

The ratio of PKC βII to ζ is also strongly correlated with tumorpresence (Table 1 and FIG. 6).

TABLE 1 Relationship between PKC βII: ζ ratio and tumor incidence. PKCβII:ζ ratio Animals with tumors 4.27 ± 2.37 p = 0.02 Animals withouttumors 0.71 ± 0.14

EXPERIMENT 2

Utilization of Isolated Fecal Poly A+ RNA to Detect Colon Cancer MarkersII

Liver fatty acid binding protein (L-FABP) and intestinal fatty acidbinding protein (I-FABP), expressed in colonocytes, are additional coloncancer biomarkers. Data indicates that expression of L-FABP and I-FABPare significantly depressed in carcinogen treated animals. FIG. 2documents a typical gel containing rapid competitive PCR products forL-FABP. The upper band represents the sample (390 base pairs), whereasthe lower band is the internal standard (336 base pairs).

EXPERIMENT 3

Human Clinical Trials Methodology

Clinical. Patients presenting for colonoscopy are individually typedas: 1) being free of colon cancer, 2) having adenomatous polyps(considered preneoplastic), or 3) having colon cancer (presentinghistological evidence of adenocarcinomas). Thirty subjects for eachgroup are recruited in order to reduce the effect of individualvariation on the analysis. The sample size is based upon testingequality of means with a α=0.05 and detecting a difference of size αwith a probability of 95% (Pearson and Hartley, 1966). To achieve thislevel of statistical power requires 26 individuals. Thus 30 patientsprotect against loss of power if a sample becomes damaged during storageor analysis. Because patients randomly present for treatment, and thedisease state will not be a controlled factor, we assume the data willbe randomly distributed among the potential population. Because patientswith cancer are the limiting factor in sample collection, the first 30individuals with cancer are those selected for inclusion in the study.In order to adjust for variation related to patient age, individualsfree of colon cancer and those with polyps are age-matched to patientswith colon cancer. Further, patients with polyps or free of pathologyare selected after a sample is collected from a cancer patient.

A patient will follow a bowel preparation schedule prior to colonoscopy.Patients will receive the Golytely™ (3-4 L, Braintree Labs, Braintree,Mass.) colonoscopic preparation. This preparation was selected becauseit preserves surface epithelial and goblet cells and has minimal effectson a variety of colon cancer risk biomarkers. At the time ofcolonoscopy, the rectal vault eluate (5-50 ml) will be suctioned throughthe scope into a disposable suction trap. The trap will be removed andits contents transferred immediately into Lysis solution (from poly A+Pure Kit, Ambion, Austin, Tex.) and placed on ice until the end of thecase (<45 min). Samples will subsequently be stored at −80° C. untiltransported to the analysis lab for further processing.

Laboratory

Samples are stored at −80° C. until being thawed on ice, and thehomogenate transferred to sterile tubes and the volume measured.Dilution buffer (Ambion Poly A+ Pure Kit) is added and the contentsmixed by inversion and then centrifuged at 4,000×g for 15 min at 4° C.Oligo dT resin is added to the sample and the supernatant is then mixedby inversion to resuspend the oligo dT resin prior to rocking the tubeon a horizontal shaker. Following centrifugation, the resin pellet isresuspended with binding buffer (Ambion Kit). The resin is thenpelleted, supernatant discarded and resulting poly A+ RNA eluted fromthe resin and used to determine biomarker prevalence (Davidson et al.,1995). The biomarkers chosen for analysis are PKC βII and PKC ζ, basedon our previous research indicating the βII isoform is positivelycorrelated with colon tumor incidence (FIG. 4A), and the ζ isoform isnegatively correlated with tumor incidence. In addition, cyclin D₁(Arber, 1996), survivin (Lacasse, 1998), cyclooxygenase type II(Kutchera, 1996), p53 (El-Mahdani, 1997), and human telomerase reversetranscriptase (hTERT) (Sumida, 1999) were selected based on the citedresearch indicating a strong correlation between mRNA expression andtumor incidence. RC-PCR (Jiang et al., 1996) is used to detect the levelof expression for each of the biomarkers.

A representative agarose gel showing quantitative RT-PCR of human PKCβII and ζ is shown in FIG. 7. The fidelity of all PCR reactions wasconfirmed by DNA sequencing (Davidson, 1994). Negative controlsprocessed without RT yielded no detectable amplified products indicatingthe absence of DNA contamination. Comparable results were obtained fromfreshly isolated fecal samples (refer to FIG. 7 for details).

Data is analyzed using the GLM models of SAS. Differences between groupsare determined by orthogonal contrasts. Data from healthy individualsare compared with those having either polyps or cancer to determine ifthe presence of the pathologies affect the relative mRNA expression forthe genes with biomarker potential. In addition, a contrast of theindividuals with polyps vs those with cancer is performed to determineif the expression changes with stage of the tumorigenic process.

EXPERIMENT 4

Detection of Fecal Protein Kinase C βII AND ζMessenger RNA Colon CancerBiomarkers

The animal use protocol conformed to NIH guidelines and was approved bythe University Animal Care Committee of Texas A&M University.Forty-eight male weanling Sprague-Dawley rats (Harlan Sprague-Dawley,Houston, Tex.) were randomly divided into two groups as previouslydescribed (Chang et al., 1997) and given two types of injection(carcinogen or saline). Animals were housed individually in suspendedcages in a temperature and humidity controlled animal facility with a 12h light/dark cycle. Food and distilled water were freely available.Forty-eight h food intakes and fecal outputs were measured during thestudy. Body weights were recorded weekly.

Carcinogen Administration and Fecal Collection

After a 2 week acclimation period, rats were given two s.c. injection ofAOM (Sigma Chemical Co., St. Louis, Mo.) at a dose of 15 mg/kg bodyweight or an equal volume of saline (one injection/week) (Chang et al.,1997). Animals were killed by CO₂ asphyxiation 36 weeks after the secondinjection. The colon was subsequently removed and the most distal fecalpellet collected. The pellet was immediately placed in Lysis solutionfor RNA isolation (Ambion Totally RNA kit, Austin, Tex.). The colon wasthen visually inspected for tumors and tumor typing was determined(Chang et al., 1997). Briefly, tissue sections were fixed in 4% bufferedformalin, embedded in paraffin, and stained with eosin and hematoxylin.Slides were then microscopically evaluated for tumors as previouslydescribed (Chang et al., 1997). Following removal of suspected tumorsfor histological evaluation, the remaining colonic sections were gentlyscraped with a microscopic slide and the mucosa used for determinationof steady-state levels of PKC isozyme mRNA. Histological evaluation ofthis method indicated that epithelial cells and lamina propria down tothe muscularis mucosa were removed (Lee et al., 1992).

RNA Isolation

Fecal poly A+ RNA was prepared as described above. Quantification offecal poly A+ RNA was performed as previously described (Davidson etal., 1995). Briefly, samples were quantitated by blotting fecal poly A+RNA onto a positively charged nylon membrane (Roche, Indianapolis,Ind.). A biotinylated oligo (dT) probe (Promega, Madison, Wis.) washybridized to the poly A+RNA followed by detection withstreptavidin-alkaline phosphatase. Dilutions of colonic musocal totalRNA of known concentration (as determined from absorbance at 260 nm)were also blotted to generate a standard curve. For concentrationcalculations, it was assumed that poly A+ RNA constitutes 3% of totalRNA.

Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Assay forNegative Controls (PKC γ and PKC βI)

Aliquots of 40 ng fecal poly A+ RNA in a 50 μl reaction were reversetranscribed to generate first strand cDNA using Superscript II reversetranscriptase (Gibco-BRL, Gaithersburg, Md.) as previously described(Davidson et al., 1995). PCR was performed using Expand High FidelityDNA polymerase (Roche, Indianapolis, Ind.). The 50 μl PCR reactionconsisted of 1× PCR buffer, 2% DMSO, 0.05 mM dNTPs, 1.5 mM MgCl₂, 20pmol each of forward and reverse primer, 2.6U Expand High Fidelity DNApolymerase and 10 μl of RT reaction. Rat brain cDNA was run as apositive control. PCR was performed using a Perkin-Elmer 2400 thermalcycler (Perkin-Elmer, Foster City, Calif.) with the followingamplification program: 15 s denaturation (94°), 15 s annealing (59° C.)and 45 s extension (74° C.) for 40 cycles. PCR products were analyzed ona 4% agarose gel followed by ethidium bromide staining. All PCR productswere sequenced to ensure the fidelity of amplification (Davidson et al.,1994). The primer pair for PKC γ was as follows (347 bp); forward,5′-TTGATGGGGAAGATGAGGAGG-3′, Sequence ID No. 1; reverse,5′-GAAATCAGCTTGGTCGATGCTG-3′, Sequence ID No. 2. The primer pair for PKCβI was as follows (639 (bp): forward, 5′-TGTGATGGAGTATGTGAACGGGGG-3′,Sequence ID No. 3; reverse, 5′-TCGAAGTTGGAGGTGTCTCGCTTG-3′, Sequence IDNo. 4.

Rapid Competitive Reverse Transcription-Polymerase Chain Reaction AssayFor Fecal and Mucosal PKC ζ and βII

Rapid competitive RT-PCR was performed in order to semi-quantitativelydetermine the PKC ζ and βII fecal and mucosal mRNA levels as previouslydescribed (Jiang et al., 1996). Using this method, relative geneexpression was determined by co-amplifying an exogenous DNA target(‘internal standard’) with a different size than the sample cDNA butwith identical 5′ and 3′ ends. This allows for competition between thesample cDNA and the internal standards for primers (Jiang et al., 1996).Internal standards were prepared as described previously (Davidson etal., 1995). Fecal poly A+ RNA was processed as described above. Inaddition, 6 μg of mucosal total RNA was reverse transcribed in a 50 μlreaction and 10 μl was amplified in the presence of either 140 fg of PKCζ internal standard or 31.2 fg PKC βII internal standard. The primerpair for the PKC ζ internal standard was (561 bp): forward,5′-CGATGGGGTGGATGGGATCAAAA-3′, Sequence ID No. 5; reverse,5′-GTATTCATGTCAGGGTTGTCTGGATTTCGGGGGCG-3′, Sequence ID No. 6, and forPKC ζ was (680 bp): forward, 5′-CGATGGGGTGGATGGGATCAAAA-3′, Sequence IDNo. 7; reverse, 5′-GTATTCATGTCAGGGTTGTCTG-3′, Sequence ID No. 8. Theprimer pair for PKC βII internal standard was (361 bp): forward,5′-TATCTGGGATGGGGTGACAACCGAGATCATTGCTTA-3′, Sequence ID No. 9; reverse,5′-CGGTCGAAGTTTTCAGCGTTTC-3′, Sequence ID No. 10. The primer pair forPKC βII was (419 bp): forward, 5′-TATCTGGGATGGGGTGACAACC-3′, Sequence IDNo. 11; reverse, 5′-CGGTCGAAGTTTTCAGCGTTTC-3′, Sequence ID NO. 12. PCRproducts were separated on a 4% agarose gel and stained with ethidiumbromide. A representative gel is shown in FIG. 3. Gels were scanned andband intensities quantitated with Biolmage software version 2.1 (AnnArbor, Mich.). The relative amount of sample mRNA was calculated bydividing the sample band intensity by the internal standard bandintensity. Specific amplification of mRNA was monitored by running PCRnegative controls consisting of tubes containing either sample RNAwithout reverse transcription, reverse transcribed sample without mimic,or mimic only. To ensure reproducibility of results, selected sampleswere amplified in duplicate. In addition, the fidelity of all PCRreactions was confirmed by DNA sequencing (Jiang et al., 1996).

Statistical Analysis

Data were analyzed to determine the effects of carcinogen and presenceof tumor using one-way ANOVA. When P-values were <0.05 for the effectsof tumor or carcinogen, total means were separated using Duncan'smultiple range test.

RESULTS

Colon Carcinoma Incidence

There was no evidence of carcinoma in any saline injected animal,whereas 64% of carcinogen injected rats had carcinomas at the time ofdeath.

Effect of Carcinogen and Presence of Tumor on Fecal and Mucosal PKCIsozyme mRNA Levels

To determine the specificity of this non-invasive procedure, PKC βI andγ primers were used as negative controls (Davidson et al., 1994;Davidson et al., 1995). No amplified products were detected after 40cycles in any fecal poly A+ or scraped colonic mucosa total RNA samples(FIG. 3, lanes 3, 4, 6 and 7). However, both isozymes were detectedusing brain total RNA (positive control, lanes 2 and 5).

PCR products for PKC βII were detected in all fecal and mucosal samples.Samples processed without reverse transcriptase were used as negativecontrols and yielded no detectable amplified products (data not shown).Using semiquantitative mimic PCR, it was determined that fecal PKC βIImRNA levels were altered by the presence of a tumor with tumor-bearinganimals having 3-fold higher (P<0.05) PKC βII expression as comparedwith animals without tumors, as seen in FIG. 4A. In contrast, there wasno effect of tumor incidence on mucosal PKC βII expression. However,there was a significant effect (P<0.05) of injection on mucosal PKC βIIexpression. Specifically, carcinogen (AOM) injection increased mucosalPKC βII mRNA expression compared with saline controls (FIG. 4B).

Colonic mucosal PKC ζ expression in rats injected with AOM was less thanhalf (P<0.05) that of saline control, as shown in FIG. 5. Since tumorincidence exerts a reciprocal effect on fecal PKC β and PKC βIIexpression, data were also expressed as the ratio between PKC βII andPKC ζ. The isozyme ratio was strongly related to tumor incidence, i.e.ratio for animals with tumors was 2.18±1.25 (n=9), animals withouttumors was 0.50±0.6 (n=26), P=0.025 (FIG. 6). These data demonstratethat PKC βII and PKC ζ may serve as non-invasive markers for developmentof colon tumors.

EXPERIMENT 5

Enhancement of Noninvasive mRNA-Gene Expression Profiling Using BiochipTechnology

mRNA isolated from feces can be utilized in combination withcomplimentary DNA (cDNA) and oligonucleotide microarray technology inorder to noninvasively determine complex patterns of gene expression,and mutations (Bowtell, 1999; Duggan, 1999; Lipshutz, 1999). Biochiptechnology is described in many publications (including Bowtell, 1999;Duggan, 1999; Lipshutz, 1999 which are incorporated herein byreference), and is known in the art. This technology allows forlarge-scale, high-throughput monitoring of gene expression patterns ofup to 40,000 genes (Bowtell, 1999; Duggan, 1999; Lipshutz, 1999).Generated data provide insight into the extent of expression differencesunderlying colonic disease, e.g., malignancy, and reveal genes that mayprove useful as diagnostic or prognostic markers.

Description of the Method

0.1-1 μg of fecal poly A+ RNA isolated from animal/human subjects aspreviously described, is processed in strict accordance to the followingprotocols or others known in the art. For example, following fecal mRNAisolation, cDNA synthesis will be performed using select primers, suchas, for example (a T7-(dT)₂₄-3′ primer:5′-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(dT)₂₄-3′) (Sequence ID No.13). Subsequently, in vitro transcription is performed to generatelabeled samples for hybridization. This technology is known in the art.cRNA fragmentation, target hybridization, fluidics station setup, probearray washing and staining, probe array scan, and initial data analysisare performed according to procedures known in the art. The precisecomposition of the probe microarray can vary depending on the specificpackage of genes being surveyed. The microarrays are currently capableof simultaneously quantitating mRNA levels (gene expression) forthousands of genes in a single experiment. Quantitative changes in mRNAexpression patterns of approximately 2-fold or greater can be detected(Bowtell, 1999; Duggan, 1999; Lipshutz, 1999). With regard tospecificity, hybridization discrimination of low abundance transcriptsis currently 1:50,000-1:100,000.

Fecal (exfoliated colonic cell) mRNA isolation methodology incombination with Biochip technology can be utilized to assay for anumber of gene expression applications. For example:

1. Tissue comparison: diseased (e.g., colon cancer, colitis) vs.unaffected colon, as a means of predicting disease onset.

2. Time point experiments: determine patient status over time.

3. Drug response in the body.

Explanation of GeneChip Probe Arrays

GeneChip probe arrays are known in the art and in essence aremanufactured using technology that combines photolithographic methodsand combinational chemistry. Tens to hundreds of thousands of differentoligonucleotide probes are synthesized, for example, in a 1.28 cm×1.28cm area on each array. Each probe type is located in a specific area onthe probe array called a probe cell. Each probe cell contains millionsof copies of a given probe. In use, biotin-labeled RNA fragments,referred to as the RNA targets, are hybridized to the probe array. Thehybridized probe array is stained with, for example, streptavidinphycoerythrin conjugate and scanned by the Hewlett-Packard (HP)GeneArray™ Scanner at the excitation wavelength of 488 nm. The amount oflight emitted at 570 nm is proportional to bound target at each locationon the probe array.

Target Preparation

Double stranded cDNA is synthesized from poly A+ messenger RNA isolatedfrom tissue or cells. An in vitro reaction is then performed to producebiotin-labeled cRNA from the cDNA. The cRNA is fragmented beforehybridization.

Target Hybridization

After the biotin-labeled cRNA is fragmented, a hybridization cocktail isprepared, which includes the fragmented cRNA, probe array controls, BSA,and herring sperm DNA. A cleanup procedure is performed on thehybridization cocktail after which approximately 200 μL is applied tothe probe array. It is then hybridized to the oligonucleotide probes onthe probe array during a 16-hour incubation at 45° C.

Probe Array Washing and Staining

Immediately following the hybridization, the hybridized probe arrayundergoes a washing and staining protocol as known in the art.

Probe Array Scan

Once the probe array has been hybridized, stained, and washed, it isscanned as known in the art.

Data Analysis

Data are analyzed using the GeneChip software available in the art. Thedata image is analyzed for probe intensities and results are reported intabular and graphical formats.

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One skilled in the art readily appreciates that the present invention iswell adapted to carry out the objectives and obtain the ends andadvantages mentioned as well as those inherent therein. Systems,biochemical compositions, treatments, methods, procedures and techniquesdescribed herein are presently representative of the preferredembodiments and are intended to be exemplary and are not intended aslimitations of the scope. Changes therein and other uses will occur tothose skilled in the art which are encompassed within the spirit of theinvention or defined by the scope of the pending claims.

12 1 21 DNA Rattus sp. 1 ttgatgggga agatgaggag g 21 2 22 DNA Rattus sp.2 gaaatcagct tggtcgatgc tg 22 3 24 DNA Rattus sp. 3 tgtgatggagtatgtgaacg gggg 24 4 24 DNA Rattus sp. 4 tcgaagttgg aggtgtctcg cttg 24 523 DNA Rattus sp. 5 cgatggggtg gatgggatca aaa 23 6 35 DNA Rattus sp. 6gtattcatgt cagggttgtc tggatttcgg gggcg 35 7 23 DNA Rattus sp. 7cgatggggtg gatgggatca aaa 23 8 22 DNA Rattus sp. 8 gtattcatgt cagggttgtctg 22 9 36 DNA Rattus sp. 9 tatctgggat ggggtgacaa ccgagatcat tgctta 3610 22 DNA Rattus sp. 10 cggtcgaagt tttcagcgtt tc 22 11 22 DNA Rattus sp.11 tatctgggat ggggtgacaa cc 22 12 22 DNA Rattus sp. 12 cggtcgaagttttcagcgtt tc 22

What is claimed is:
 1. A method for non-invasively determining theexpression of PKC isozymes in colonocytes of a patient comprising:directly isolating from said patient polyA+RNA from feces, containingsloughed colonocytes; and assaying the isolated polyA+RNA anddetermining the level, in the isolated A+RNA, of mRNA encoding PKCisozymes wherein the PKC isozyme are PKC ζ and PKC βII.
 2. The method ofclaim 1, wherein the ratio of expression of PKC βII to PKC ζ isdetermined.
 3. The method of claim 2, further comprising the step ofcomparing the ratio of expression of PKC βII to PKC ζ in said patientwith similarly determined ratios of PKC βII to PKC ζ in other patientswith known conditions.
 4. The method of claim 3, wherein the level ofexpression of PKC βII to PKC ζ in said patient is compared withsimilarly determined ratios of PKC βII to PKC ζ in at least two otherpatients, one with colon cancer and one without colon cancer.
 5. Themethod of claim 2, wherein the level of PKC ζ is determined using theprimer pair having Sequence ID Numbers 7 and 8, and the level of PKC βIIis determined using the primer pair selected from the group consistingof the primers having Sequence ID Numbers 11, and
 12. 6. A method fornon-invasively detecting colonic biomarkers in a patient using fecalmessenger RNA comprising: directly isolating, from said patient,polyA+RNA from feces containing sloughed colonocytes; and assaying theisolated polyA+RNA and determining the level, in the isolated polyA+RNA,of mRNA encoding colonic biomarkers wherein the colonic biomarker arePKC ζ and PKC βII.
 7. The method of claim 6, wherein the ratio ofexpression of PKC βII to PKC ζ is determined.
 8. The method of claim 7,further comprising the step of comparing the ratio of expression of PKCβII to PKC ζ in said patient with similarly determined ratios of PKC βIIto PKC ζ in at least two other patients, one with colon cancer and onewithout colon cancer.
 9. The method of claim 6, wherein the level of PKCζ is determined using at least one primer selected from the groupconsisting of the primers having Sequence ID Numbers 5, 6, 7, and 8, andthe level of PKC βII is determined using at least one primer selectedfrom the group consisting of the primers having Sequence ID Numbers 9,10, 11, and
 12. 10. A method for non-invasively screening for coloncancer in a patient comprising: detecting the expression of PKC ζ andPKC βII in sloughed colonocytes in said patient's feces; and correlatingthe expression of PKC ζ and PKC βII with the presence or absence ofcolon cancer in said patient.
 11. The method of claim 10, wherein theratio of expression of PKC βII to PKC ζ is determined.
 12. The method ofclaim 10, further comprising the step of comparing the ratio ofexpression of PKC βII to PKC ζ in said patient with similarly determinedratios of PKC βII to PKC ζ in at least two other patients, one withcolon cancer and one without colon cancer.
 13. The method of claim 10,wherein the level of PKC ζ is determined using at least one primerselected from the group consisting of the primers having Sequence IDNumbers 5, 6, 7, and 8, and the level of PKC βII is determined using atleast one primer selected from the group consisting of the primershaving Sequence ID Numbers 9, 10, 11, and 12.